Dry stable formation to produce microbubble suspension for ultrasound

ABSTRACT

Gas or air filled microbubble suspensions in aqueous phases usable as imaging contrast agents in ultrasonic echography. They contain laminarized surfactants and, optionally, hydrophilic stabilizers. The laminarized surfactants can be in the form of liposomes. The suspensions are obtained by exposing the laminarized surfactants to air or a gas before or after admixing with an aqueous phase.

This is a Rule 60 Division of application Ser. No. 08/456,385 U.S. Pat.No. 5,658,551 filed 1 June 1995 which is a division of Ser. No.08/315,347 filed Sep. 30, 1994 U.S. Pat. No. 5,531,980 which is adivision of Ser. No. 08/128,540 filed Sep. 29, 1993 now U.S. Pat. No.5,380,519 which is a 371 of PCT/EP91/00620 filed Apr. 2, 1991 and is adivision of Ser. No. 07/775,989, filed Nov. 20, 1991, now U.S. Pat. No.5,271,928.

The present invention concerns media adapted for injection into livingbodies, e.g. for the purpose of ultrasonic echography and, moreparticularly, injectable liquid compositions comprising microbubbles ofair or physiologically acceptable gases as stable dispersions orsuspensions in an aqueous liquid carrier. These compositions are mostlyusable as contrast agents in ultrasonic echography to image the insideof blood-stream vessels and other cavities of living beings, e.g. humanpatients and animals. Other uses however are also contemplated asdisclosed hereafter.

The invention also comprises dry compositions which, upon admixing withan aqueous carrier liquid, will generate the foregoing sterilesuspension of microbubbles thereafter usable as contrast agent forultrasonic echography and other purposes.

It is well known that microbodies like microspheres or microglobules ofair or a gas, e.g. microbubbles or microballoons, suspended in a liquidare exceptionally efficient ultrasound reflectors for echography. Inthis disclosure the term of "microbubble" specifically designates air orgas globules in suspension in a liquid which generally results from theintroduction therein of air or a gas in divided form, the liquidpreferably also containing surfactants or tensides to control thesurface properties thereof and the stability of the bubbles. Morespecifically, one may consider that the internal volume of themicrobubbles is limited by the gas/liquid interface, or in other words,the microbubbles are only bounded by a rather evanescent envelopeinvolving the molecules of the liquid and surfactant loosely bound atthe gas to liquid junction boundary.

In contrast, the term of "microcapsule" or "microballoon" designatespreferably air or gas bodies with a material boundary or envelope formedof molecules other than that of the liquid of suspension, e.g. a polymermembrane wall. Both microbubbles and microballoons are useful asultrasonic contrast agents. For instance injecting into the blood-streamof living bodies suspensions of gas microbubbles or microballoons (inthe range of 0.5 to 10 μm) in a carrier liquid will strongly reinforceultrasonic echography imaging, thus aiding in the visualization ofinternal organs. Imaging of vessels and internal organs can stronglyhelp in medical diagnosis, for instance for the detection ofcardiovascular and other diseases.

The formation of suspensions of microbubbles in an injectable liquidcarrier suitable for echography can follow various routes. For instancein DE-A- 3529195 (Max-Planck Gesell.), there is disclosed a techniquefor generating 0.5-50 μm bubbles in which an aqueous emulsified mixturecontaining a water soluble polymer, an oil and mineral salts is forcedback and forth, together with a small amount of air, from one syringeinto another through a small opening. Here, mechanical forces areresponsible for the formation of bubbles in the liquid.

M. W. Keller et al. (J. Ultrasound Med. 5 (1986), 439-8) have reportedsubjecting to ultrasonic cavitation under atmospheric pressure solutionscontaining high concentrations of solutes such as dextrose,Renografin-76, Iopamidol (an X-ray contrast agent), and the like. Therethe air is driven into the solution by the energy of cavitation.

Other techniques rely on the shaking of a carrier liquid in which aircontaining microparticles have been incorporated, said carrier liquidusually containing, as stabilizers, viscosity enhancing agents, e.g.water soluble polypeptides or carbohydrates and/or surfactants. It iseffectively admitted that the stability of the microbubbles againstdecay or escape to the atmosphere is controlled by the viscosity andsurface properties of the carrier liquid. The air or gas In themicroparticles can consist of inter-particle or intra-crystallineentrapped gas, as well as surface adsorbed gas, or gas produced byreactions with the carrier liquid, usually aqueous. All this is fullydescribed for instance in EP-A- 52.575 (Ultra Med. Inc.) In which thereare used aggregates of 1-50 μm particles of carbohydrates (e.g.galactose, maltose, sorbitol, gluconic acid, sucrose, glucose and thelike) in aqueous solutions of glycols or polyglycols, or other watersoluble polymers.

Also, in EP-A- 123.235 and 122.624 (Schering, see also EP-A- 320.433)use is made of air trapped in solids. For instance, 122.624 claims aliquid carrier contrast composition for ultrasonic echography containingmicroparticles of a solid surfactant, the latter being optionallycombined with microparticles of a non-surfactant. As explained in thisdocument, the formation of air bubbles in the solution results from therelease of the air adsorbed on the surface of the particles, or trappedwithin the particle lattice, or caught between individual particles,this being so when the particles are agitated with the liquid carrier.

EP-A- 131.540 (Schering) also discloses the preparation of microbubblessuspensions in which a stabilized injectable carrier liquid, e.g. aphysiological aqueous solution of salt, or a solution of a sugar likemaltose, dextrose, lactose or galactose, without viscosity enhancer, ismixed with microparticles (in the 0.1 to 1 μm range) of the same sugarscontaining entrapped air. In order that the suspension of bubbles candevelop within the liquid carrier, the foregoing documents recommendthat both liquid and solid components be violently agitated togetherunder sterile conditions; the agitation of both components together isperformed for a few seconds and, once made, the suspension must then beused immediately, i.e. it should be injected within 5-10 minutes forechographic measurements; this indicates that the bubbles in thesuspensions are not longlived and one practical problem with the use ofmicrobubbles suspensions for injection is lack of stability with time.The present invention fully remedies this drawback.

In U.S. Pat. No. 4,466,442 (Schering), there is disclosed a series ofdifferent techniques for producing suspensions of gas microbubbles in aliquid carrier using (a) a solution of a tenside (surfactant) in acarrier liquid (aqueous) and (b) a solution of a viscosity enhancer asstabilizer. For generating the bubbles, the techniques used thereinclude forcing at high velocity a mixture of (a), (b) and air through asmall aperture; or injecting (a) into (b) shortly before use togetherwith a physiologically acceptable gas; or adding an acid to (a) and acarbonate to (b), both components being mixed together just before useand the acid reacting with the carbonate to generate CO₂ bubbles; oradding an over-pressurized gas to a mixture of (a) and (b) understorage, said gas being released into microbubbles at the time when themixture is used for injection.

The tensides used in component (a) of U.S. Pat. No. 4,466,442 compriselecithins; esters and ethers of fatty acids and fatty alcohols withpolyoxyethylene and polyoxyethylated polyols like sorbitol, glycols andglycerol, cholesterol; and polyoxy-ethylene-polyoxypropylene polymers.The viscosity raising and stabilizing compounds include for instancemono- and polysaccharides (glucose, lactose, sucrose, dextran,sorbitol); polyols, e.g. glycerol, polyglycols; and polypeptides likeproteins, gelatin, oxypolygelatin, plasma protein and the like.

In a typical preferred example of this document, equivalent volumes of(a) a 0.5% by weight aqueous solution of Pluronic® F-68 (apolyoxypropylene-polyoxyethylene polymer) and (b) a 10% lactose solutionare vigorously shaken together under sterile conditions (closed vials)to provide a suspension of microbubbles ready for use as an ultrasoniccontrast agent and lasting for at least 2 minutes. About 50% of thebubbles had a size below 50 μm.

Although the achievements of the prior art have merit, they suffer fromseveral drawbacks which strongly limit their practical use by doctorsand hospitals, namely their relatively short life-span (which makes testreproducibility difficult), relative low initial bubble concentration(the number of bubbles rarely exceeds 10⁴ -10⁵ bubbles/ml and the countdecreases rapidly with time) and poor reproducibility of the initialbubble count from test to test (which also makes comparisons difficult).Also it is admitted that for efficiently imaging certain organs, e.g.the left heart, bubbles smaller than 50 μm, preferably in the range of0.5-10 μm, are required: with longer bubbles, there are risks of clotsand consecutive emboly.

Furthermore, the compulsory presence of solid microparticles or highconcentrations of electrolytes and other relatively inert solutes in thecarrier liquid may be undesirable physiologically In some cases.Finally, the suspensions are totally unstable under storage and cannotbe marketed as such; hence great skill is required to prepare themicrobubbles at the right moment just before use.

Of course there exists stable suspensions of microcapsules, i.e.microballoons with a solid, air-sealed rigid polymeric membrane whichperfectly resist for long storage periods in suspension, which have beendeveloped to remedy this shortcoming (see for instance K. J. Widder,EP-A- 324.938); however the properties of microcapsules in which a gasis entrapped inside solid membrane vesicles essentially differ from thatof the gas microbubbles of the present invention and belong to adifferent kind of art; for instance while the gas microbubbles discussedhere will simply escape or dissolve in the blood-stream when thestabilizers in the carrier liquid are excreted or metabolized, the solidpolymer material forming the walls of the aforementioned micro-balloonsmust eventually be disposed of by the organism being tested which mayimpose a serious afterburden upon it. Also capsules with solid,non-elastic membrane may break irreversibly under variations ofpressure.

The composition of the present invention, as defined in claim 1, fullyremedies the aforementioned pitfalls.

The term "lamellar form" defining the condition of at least a portion ofthe surfactant or surfactants of the present composition indicates thatthe surfactants, in strong contrast with the microparticles of the priorart (for instance EP-A-123.235), are in the form of thin films involvingone or more molecular layers (in laminate form). Converting film formingsurfactants into lamellar form can easily be done for instance by highpressure homogenization or by sonication under acoustical or ultrasonicfrequencies. In this connection, it should be pointed out that theexistence of liposomes is a well known and useful illustration of casesin which surfactants, more particularly lipids, are in lamellar form.

Liposome solutions are aqueous suspensions of microscopic vesicles,generally spherically shaped, which hold substances encapsulatedtherein. These vesicles are usually formed of one or more concentricallyarranged molecular layers (lamellae) of amphipatic compounds, i.e.compounds having a lipophobic hydrophilic moiety and a lipophilichydrophobic moiety. See for instance "Liposome Methodology", Ed. L. D.Leserman et al, Inserm 136, 2-8 May 1982). Many surfactants or tensides,including lipids, particularly phospholipids, can be laminarized tocorrespond to this kind of structure. In this invention, one preferablyuses the lipids commonly used for making liposomes, for instance thelecithins and other tensides disclosed in more detail hereafter, butthis does in no way preclude the use of other surfactants provided theycan be formed into layers or films.

It is important to note that no confusion should be made between thepresent invention and the disclosure of Ryan (U.S. Pat. No. 4,900,540)reporting the use of air or gas filled liposomes for echography. In thismethod Ryan encapsulates air or a gas within liposomic vesicles; inembodiments of the present invention microbubbles or air or a gas areformed in a suspension of liposomes (i.e. liquid filled liposomes) andthe liposomes apparently stabilize the microbubbles. In Ryan, the air isinside the liposomes, which means that within the bounds of thepresently used terminology, the air filled liposomes of Ryan belong tothe class of microballoons and not to that of the microbubbles of thepresent invention.

Practically, to achieve the suspensions of microbubbles according to theinvention, one may start with liposomes suspensions or solutionsprepared by any technique reported in the prior art, with the obviousdifference that in the present case the liposomic vesicles arepreferably "unloaded", i.e. they do not need to keep encapsulatedtherein any foreign material other than the liquid of suspension as isnormally the object of classic liposomes. Hence, preferably, theliposomes of the present invention will contain an aqueous phaseidentical or similar to the aqueous phase of the solution itself. Thenair or a gas is introduced into the liposome solution so that asuspension of microbubbles will form, said suspension being stabilizedby the presence of the surfactants in lamellar form. Notwithstanding,the material making the liposome walls can be modified within the scopeof the present invention, for instance by covalently grafting thereonforeign molecules designed for specific purposes as will be explainedlater.

The preparation of liposome solutions has been abundently discussed inmany publications, e.g. U.S. Pat. No. 4,224,179 and WO-A-88/09165 andall citations mentioned therein. This prior art is used here asreference for exemplifying the various methods suitable for convertingfilm forming tensides into lamellar form. Another basic reference by M.C. Woodle and D. Papahadjopoulos is found in "Methods in Enzymology" 171(1989), 193.

For instance, in a method disclosed in D. A. Tyrrell et al, Biochimica &Biophysica Acta 457 (1976), 259-302, a mixture of a lipid and an aqueousliquid carrier is subjected to violent agitation and thereaftersonicated at acoustic or ultrasonic frequencies at room or elevatedtemperature. In the present invention, it has been found that sonicationwithout agitation is convenient. Also, an apparatus for makingliposomes, a high pressure homogenizer such as the Microfluidizer®,which can be purchased from Microfluidics Corp., Newton, Mass. 02164USA, can be used advantageously. Large volumes of liposome solutions canbe prepared with this apparatus under pressures which can reach 600-1200bar.

In another method, according to the teaching of GB-A-2,134,869 (Squibb),microparticles (10 μm or less) of a hydrosoluble carrier solid (NaCl,sucrose, lactose and other carbohydrates) are coated with an amphipaticagent; the dissolution of the coated carrier in an aqueous phase willyield liposomic vesicles. In GB-A- 2,135,647 insoluble particles, e.g.glass or resin microbeads are coated by moistening in a solution of alipid in an organic solvent followed by removal of the solvent byevaporation. The lipid coated microbeads are thereafter contacted withan aqueous carrier phase, whereby liposomic vesicles will form in thatcarrier phase.

The introduction of air or gas into a liposome solution in order to formtherein a suspension of microbubbles can be effected by usual means,inter alia by injection, that is, forcing said air or gas through tinyorifices into the liposome solution, or simply dissolving the gas in thesolution by applying pressure and thereafter suddenly releasing thepressure. Another way is to agitate or sonicate the liposome solution inthe presence of air or an entrappable gas. Also one can generate theformation of a gas within the solution of liposomes itself, for instanceby a gas releasing chemical reaction, e.g. decomposing a dissolvedcarbonate or bicarbonate by acid. The same effect can be obtained bydissolving under pressure a low boiling liquid, for instance butane, inthe aqueous phase and thereafter allowing said liquid to boll bysuddenly releasing the pressure.

Notwithstanding, an advantageous method is to contact the dry surfactantin lamellar or thin film form with air or an adsorbable or entrappablegas before introducing said surfactant into the liquid carrier phase. Inthis regard, the method can be derived from the technique disclosed inGB-A-2,135,647, i.e. solid microparticles or beads are dipped in asolution of a film forming surfactant (or mixture of surfactants) in avolatile solvent, after which the solvent is evaporated and the beadsare left in contact with air (or an adsorbable gas) for a timesufficient for that air to become superficially bound to the surfactantlayer. Thereafter, the beads coated with air filled surfactant are putinto a carrier liquid, usually water with or without additives, wherebyair bubbles will develop within the liquid by gentle mixing, violentagitation being entirely unnecessary. Then the solid beads can beseparated, for instance by filtration, from the microbubble suspensionwhich is remarkably stable with time.

Needless to say that, instead of insoluble beads or spheres, one may useas supporting particles water soluble materials like that disclosed inGB-A- 2,134,869 (carbohydrates or hydrophilic polymers), whereby saidsupporting particles will eventually dissolve and final separation of asolid becomes unnecessary. Furthermore in this case, the material of theparticles can be selected to eventually act as stabilizer or viscosityenhancer wherever desired.

In a variant of the method, one may also start with dehydratedliposomes, i.e. liposomes which have been prepared normally by means ofconventional techniques in the form of aqueous solutions and thereafterdehydrated by usual means, e.g. such as disclosed in U.S. Pat. No.4,229,360 also incorporated herein as reference. One of the methods fordehydrating liposomes recommended in this reference is freeze-drying(lyophilization), i.e. the liposome solution is frozen and dried byevaporation (sublimation) under reduced pressure. Prior to effectingfreeze-drying, a hydrophilic stabilizer compound is dissolved in thesolution, for instance a carbohydrate like lactose or sucrose or ahydrophilic polymer like dextran, starch, PVP, PVA and the like. This isuseful in the present invention since such hydrophilic compounds alsoaid in homogenizing the microbubbles size distribution and enhancestability under storage. Actually making very dilute aqueous solutions(0.1-10% by weight) of freeze-dried liposomes stabilized with, forinstance, a 5:1 to 10:1 weight ratio of lactose to lipid enables toproduce aqueous microbubbles suspensions counting 10⁸ -10⁹microbubbles/ml (size distribution mainly 0.5-10 μm) which are stablefor at least a month (and probably much longer) without significantobservable change. And this is obtained by simple dissolution of theair-stored dried liposomes without shaking or any violent agitation.Furthermore, the freeze-drying technique under reduced pressure is veryuseful because it permits, after drying, to restore the pressure abovethe dried liposomes with any entrappable gas, i.e. nitrogen, CO₂, argon,methane, FREON, etc., whereby after dissolution of the liposomesprocessed under such conditions suspensions of microbubbles containingthe above gases are obtained.

Microbubbles suspensions formed by applying gas pressure on a dilutesolution of laminated lipids in water (0.1-10% by weight) and thereaftersuddenly releasing the pressure have an even higher bubbleconcentration, e.g. in the order of 10¹⁰ -10¹¹ bubbles/ml. However, theaverage bubble size is somewhat above 10 μm, e.g. in the 10-50 μm range.In this case, bubble size distribution can be narrowed by centrifugationand layer decantation.

The tensides or surfactants which are convenient in this invention canbe selected from all amphipatic compounds capable of forming stablefilms in the presence of water and gases. The preferred surfactantswhich can be laminarized include the lecithins (phosphatidyl-choline)and other phospholipids, inter alia phosphatidic acid (PA),phosphatidyl-inositol phosphatidyl-ethanolamine (PE),phosphatidyl-serine (PS), phosphatidyl-glycerol (PG), cardiolipin (CL),sphingomyelins, the plasmogens, the cerebrosides, etc. Examples ofsuitable lipids are the phospholipids in general, for example, naturallecithins, such as egg lecithin or soya bean lecithin, or syntheticlecithins such as saturated synthetic lecithins, for example,dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline ordistearoyl phosphatidyl choline or unsaturated synthetic lecithins, suchas dioleyl phosphatidyl choline or dilinoleyl phosphatidyl choline, withegg lecithin or soya bean lecithin being preferred. Additives likecholesterol and other substances (see below) can be added to one or moreof the foregoing lipids in proportions ranging from zero to 50% byweight.

Such additives may include other surfactants that can be used inadmixture with the film forming surfactants and most of which arerecited in the prior art discussed in the introduction of thisspecification. For instance, one may cite free fatty acids, esters offatty acids with polyoxyalkylene compounds like polyoxypropylene glycoland polyoxyethylene glycol; ethers of fatty alcohols withpolyoxyalkylene glycols; esters of fatty acids with polyoxyalkylatedsorbitan; soaps; glycerol-polyalkylene stearate;glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyakylated; mono-, di- and triglycerides of saturated orunsaturated fatty acids; glycerides of soya-oil and sucrose. The amountof the non-film forming tensides or surfactants can be up to 50% byweight of the total amount of surfactants in the composition but ispreferably between zero and 30%.

The total amount of surfactants relative to the aqueous carrier liquidis best in the range of 0.01 to 25% by weight but quantities in therange 0.5-5% are advantageous because one always tries to keep theamount of active substances in an injectable solution as low aspossible, this being to minimize the introduction of foreign materialsinto living beings even when they are harmless and physiologicallycompatible.

Further optional additives to the surfactants include:

a) substances which are known to provide a negative charge on liposomes,for example, phosphatidic acid, phosphatidyl-glycerol or dicetylphosphate;

b) substances known to provide a positive charge, for example, stearylamine, or stearyl amine acetate;

c) substances known to affect the physical properties of the lipid filmsIn a more desirable way: for example, capro-lactam and/or sterols suchas cholesterol, ergosterol, phytosterol, sitosterol, sitosterolpyroglutamate, 7-dehydro-cholesterol or lanosterol, may affect lipidfilms rigidity;

d) substances known to have antioxidant properties to improve thechemical stability of the components in the suspensions, such astocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene.

The aqueous carrier in this invention is mostly water with possiblysmall quantities of physiologically compatible liquids such asisopropanol, glycerol, hexanol and the like (see for instance EP-A-52.575). In general the amount of the organic hydrosoluble liquids willnot exceed 5-10% by weight.

The present composition may also contain dissolved or suspended thereinhydrophilic compounds and polymers defined generally under the name ofviscosity enhancers or stabilizers. Although the presence of suchcompounds is not compulsory for ensuring stability to the air or gasbubbles with time in the present dispersions, they are advantageous togive some kind of "body" to the solutions. When desired, the upperconcentrations of such additives when totally innocuous can be veryhigh, for instance up to 80-90% by weight of solution with Iopamidol andother iodinated X-ray contrast agents. However with other viscosityenhancers like for instance sugars, e.g. lactose, sucrose, maltose,galactose, glucose, etc. or hydrophilic polymers like starch, dextran,polyvinyl alcohol, polyvinyl-pyrrolidone, dextrin, xanthan or partlyhydrolyzed cellulose oligomers, as well as proteins and polypeptides,the concentrations are best between about 1 and 40% by weight, a rangeof about 5-20% being preferred.

Like in the prior art, the injectable compositions of this invention canalso contain physiologically acceptable electrolytes; an example is anisotonic solution of salt.

The present invention naturally also includes dry storable pulverulentblends which can generate the present microbubble containing dispersionsupon simple admixing with water or an aqueous carrier phase. Preferablysuch dry blends or formulations will contain all solid ingredientsnecessary to provide the desired microbubbles suspensions upon thesimple addition of water, i.e. principally the surfactants in lamellarform containing trapped or adsorbed therein the air or gas required formicrobubble formation, and accessorily the other non-film formingsurfactants, the viscosity enhancers and stabilizers and possibly otheroptional additives. As said before, the air or gas entrappment by thelaminated surfactants occurs by simply exposing said surfactants to theair (or gas) at room or super-atmospheric pressure for a time sufficientto cause said air or gas to become entrapped within the surfactant. Thisperiod of time can be very short, e.g. in the order of a few seconds toa few minutes although over-exposure, i.e. storage under air or under agaseous atmosphere is in no way harmful. What is important is that aircan well contact as much as possible of the available surface of thelaminated surfactant, i.e. the dry material should preferably be in a"fluffy" light flowing condition. This is precisely this condition whichresults from the freeze-drying of an aqueous solution of liposomes andhydrophilic agent as disclosed in U.S. Pat. No. 4,229,360.

In general, the weight ratio of surfactants to hydrophilic viscosityenhancer in the dry formulations will be in the order of 0.1:10 to 10:1,the further optional ingredients, if any, being present in a ratio notexceeding 50% relative to the total of surfactants plus viscosityenhancers.

The dry blend formulations of this invention can be prepared by verysimple methods. As seen before, one preferred method is to first preparean aqueous solution in which the film forming lipids are laminarized,for instance by sonication, or using any conventional technique commonlyused in the liposome field, this solution also containing the otherdesired additives, i.e. viscosity enhancers, non-film formingsurfactants, electrolyte, etc., and thereafter freeze drying to a freeflowable powder which is then stored in the presence of air or anentrappable gas.

The dry blend can be kept for any period of time in the dry state andsold as such. For putting it into use, i.e. for preparing a gas or airmicrobubble suspension for ultrasonic imaging, one simply dissolves aknown weight of the dry pulverulent formulation in a sterile aqueousphase, e.g. water or a physiologically acceptable medium. The amount ofpowder will depend on the desired concentration of bubbles in theinjectable product, a count of about 10⁸ -10⁹ bubbles/ml being generallythat from making a 5-20% by weight solution of the powder in water. Butnaturally this figure is only indicative, the amount of bubbles beingessentially dependent on the amount of air or gas trapped duringmanufacture of the dry powder. The manufacturing steps being undercontrol, the dissolution of the dry formulations will providemicrobubble suspensions with well reproducible counts.

The resulting microbubble suspensions (bubble in the 0.5-10 μm range)are extraordinarily stable with time, the count originally measured atstart staying unchanged or only little changed for weeks and evenmonths; the only observable change is a kind of segregation, the largerbubbles (around 10 μm) tending to rise faster than the small ones.

It has also been found that the microbubbles suspensions of thisinvention can be diluted with very little loss in the number ofmicrobubbles to be expected from dilution, i.e. even in the case of highdilution ratios, e.g. 1/10² to 1/10⁴, the microbubble count reductionaccurately matches with the dilution ratio. This indicates that thestability of the bubbles depends on the surfactant in lamellar formrather than on the presence of stabilizers or viscosity enhancers likein the prior art. This property is advantageous in regard to imagingtest reproducibility as the bubbles are not affected by dilution withblood upon injection into a patient.

Another advantage of the bubbles of this invention versus themicrocapsules of the prior art surrounded by a rigid but breakablemembrane which may irreversibly fracture under stress is that when thepresent suspensions are subject to sudden pressure changes, the presentbubbles will momentarily contract elastically and then resume theiroriginal shape when the pressure is released. This is important inclinical practice when the microbubbles are pumped through the heart andtherefore are exposed to alternating pressure pulses.

The reasons why the microbubbles in this invention are so stable are notclearly understood. Since to prevent bubble escape the buoyancy forcesshould equilibrate with the retaining forces due to friction, i.e. toviscosity, it is theorized that the bubbles are probably surrounded bythe laminated surfactant. Whether this laminar surfactant is in the formof a continuous or discontinuous membrane, or even as closed spheresattached to the microbubbles, is for the moment unknown but underinvestigation. However the lack of a detailed knowledge of the phenomenapresently involved does not preclude full industrial operability of thepresent invention.

The bubble suspensions of the present invention are also useful in othermedical/diagnostic applications where it is desirable to target thestabilized microbubbles to specific sites in the body following theirinjection, for instance to thrombi present in blood vessels, toatherosclerotic lesions (plaques) in arteries, to tumor cells, as wellas for the diagnosis of altered surfaces of body cavities, e.g.ulceration sites in the stomach or tumors of the bladder. For this, onecan bind monoclonal antibodies tailored by genetic engineering, antibodyfragments or polypeptides designed to mimic antibodies, bioadhesivepolymers, lectins and other site-recognizing molecules to the surfactantlayer stabilizing the microbubbles. Thus monoclonal antibodies can bebound to phospholipid bilayers by the method described by L. D.Leserman, P. Machy and J. Barbet ("Liposome Technology vol. III" p. 29ed. by G. Gregoriadis, CRC Press 1984). In another approach a palmitoylantibody is first synthesized and then incorporated in phospholipidbilayers following L. Huang, A. Huang and S. J. Kennel ("LiposomeTechnology vol. III" p. 51 ed. by G. Gregoriadis, CRC Press 1984).Alternatively, some of the phospholipids used in the present inventioncan be carefully selected in order to obtain preferential uptake inorgans or tissues or increased half-life in blood. Thus GM1gangliosides--or phosphatidylinositol-containing liposomes, preferablyin addition to cholesterol, will lead to increased half-lifes in bloodafter intravenous administration in analogy with A. Gabizon, D.Papahadjopoulos, Proc. Natl Acad. Sci USA 85 (1988) 6949.

The gases in the microbubbles of the present invention can include, inaddition to current innocuous physiologically acceptable gases like CO₂,nitrogen, N₂ O, methane, butane, FREON and mixtures thereof, radioactivegases such as ¹³³ Xe or ⁸¹ Kr are of particular interest in nuclearmedicine for blood circulation measurements, for lung scintigraphy etc.

The following Examples illustrate the invention on a practical standpoint.

Echogenic measurements

Echogenicity measurements were performed in a pulse--echo system made ofa plexiglas specimen bolder (diameter 30 mm) and a transducer holderimmersed in a constant temperature water bath, a pulser-receiver(Accutron M3010S) with for the receiving part an external pre-amplifierwith a fixed gain of 40 dB and an internal amplifier with adjustablegain from -40 to ×40 dB. A 10 MHz low-pass filter was inserted in thereceiving part to improve the signal to noise ratio. The A/D board inthe IBM PC was a Sonotek STR 832. Measurements were carried out at 2.25,3.5, 5 and 7.5 MHz.

EXAMPLE 1

A liposome solution (50 mg lipids per ml) was prepared in distilledwater by the REV method (see F. Szoka Jr. and D. Papahadjopoulos, Proc.Natl. Acad. Sci. USA 75 (1978) 4194) using hydrogenated soya lecithin(NC 95 H, Nattermann Chemie, Koln, W. Germany) and dicetylphosphate in amolar ratio 9/1. This liposome preparation was extruded at 65° C. (tocalibrate the vesicle size) through a 1 μm polycarbonate filter(Nucleopore). Two ml of this solution were admixed with 5 ml of a 75%iopamidol solution in water and 0.4 ml of air and the mixture was forcedback and forth through a two syringe system as disclosed inDE-A-3529195, while maintaining continuously a slight over-pressure.This resulted in the formation of a suspension of microbubbles of air inthe liquid (10⁵ -10⁶ bubbles per ml, bubble size 1-20 μm as estimated bylight microscopy) which was stable for several hours at roomtemperature. This suspension gave a strong echo signal when tested byultrasonic echography at 7.5, 5, 3.5 and 2.25 MHz.

EXAMPLE 2

A distilled water solution (100 ml) containing by weight 2% ofhydrogenated soya lecithin and dicetylphosphate in a 9/1 molar ratio wassonicated for 15 min at 60-65° C. with a Branson probe sonifier (Type250).

After cooling, the solution was centrifuged for 15 min at 10,000 g andthe supernatant was recovered and lactose added to make a 7.5% b. w.solution. The solution was placed in a tight container in which apressure of 4 bar of nitrogen was established for a few minutes whileshaking the container. Afterwards, the pressure was released suddenlywhereby a highly concentrated bubble suspension was obtained (10¹⁰ -10¹¹bubbles/ml). The size distribution of the bubbles was however wider thanin Example 1, i.e. from about 1 to 50 μm. The suspension was very stablebut after a few days a segregation occurred in the standing phase, thelarger bubbles tending to concentrate in the upper layers of thesuspension.

EXAMPLE 3

Twenty g of glass beads (diameter about 1 mm) were immersed into asolution of 100 mg of dipalmitoylphosphatidylcholine (Fluka A. G. Buchs)in 10 ml of chloroform. The beads were rotated under reduced pressure ina rotating evaporator until all CHCl₃ had escaped. Then the beads werefurther rotated under atmospheric pressure for a few minutes and 10 mlof distilled water were added. The beads were removed and a suspensionof air microbubbles was obtained which was shown to contain about 10⁶bubbles/ml after examination under the microscope. The average size ofthe bubbles was about 3-5 μm. The suspension was stable for several daysat least.

EXAMPLE 4

A hydrogenated soya lecithin/dicetylphosphate suspension in water waslaminarized using the REV technique as described in Example 1. Two ml ofthe liposome preparation were added to 8 ml of 15% maltose solution indistilled water. The resulting solution was frozen at -30° C., thenlyophilized under 0.1 Torr. Complete sublimation of the ice was obtainedin a few hours. Thereafter, air pressure was restored in the evacuatedcontainer so that the lyophilized powder became saturated with air in afew minutes.

The dry powder was then dissolved in 10 ml of sterile water under gentlemixing, whereby a microbubble suspension (10⁸ -10⁹ microbubbles per ml,dynamic viscosity (<20 mPa·s) was obtained. This suspension containingmostly bubbles in the 1-5 μm range was stable for a very long period, asnumerous bubbles could still be detected after 2 months standing. Thismicrobubble suspension gave a strong response in ultrasonic echography.If in this example the solution is frozen by spraying in air at -30 to-70° C. to obtain a frozen snow instead of a monolithic block and thesnow is then evaporated under vacuum, excellent results are obtained.

EXAMPLE 5

Two ml samples of the liposome solution obtained as described in Example4 were mixed with 10 ml of an 5% aqueous solution of gelatin (sample5A), human albumin (sample 5B) dextran (sample 5C) and iopamidol (sample5D). All samples were lyophilized. After lyophilization and introductionof air, the various samples were gently mixed with 20 ml of sterilewater. In all cases, the bubble concentration was above 10⁸ bubbles perml and almost all bubbles were below 10 μm. The procedure of theforegoing Example was repeated with 9 ml of the liposome preparation(450 mg of lipids) and only one ml of a 5% human albumin solution. Afterlyophilization, exposure to air and addition of sterile water (20 ml),the resulting solution contained 2 ×10⁸ bubbles per ml, most of thembelow 10 μm.

EXAMPLE 6

Lactose (500 mg), finely milled to a particle size of 1-3 μm, wasmoistened with a chloroform (5 ml) solution of 100 mg ofdimyristoylphosphatidylcholine/cholesterol/dipalmitoylphosphatidic acid(from Fluka) in a molar ratio of 4:1:1 and thereafter evaporated undervacuum in a rotating evaporator. The resulting free flowing white powderwas rotated a few minutes under nitrogen at normal pressure andthereafter dissolved in 20 ml of sterile water. A microbubble suspensionwas obtained with about 10⁵ -10⁶ microbubbles per ml in the 1-10 μm sizerange as ascertained by observation under the microscope. In thisExample, the weight ratio of coated surfactant to water-soluble carrierwas 1:5. Excellent results (10⁷ -10⁸ microbubbles/ml) are also obtainedwhen reducing this ratio to lower values, i.e. down to 1:20, which willactually increase the surfactant efficiency for the intake of air, thatis, this will decrease the weight of surfactant necessary for producingthe same bubble count.

EXAMPLE 7

An aqueous solution containing 2% of hydrogenated soya lecithin and 0.4%of Pluronic® F68 (a non ionic polyoxyethylene-polyoxypropylene copolymersurfactant) was sonicated as described in Example 2. After cooling andcentrifugation, 5 ml of this solution were added to 5 ml of a 15%maltose solution in water. The resulting solution was frozen at -30° C.and evaporated under 0.1 Torr. Then air pressure was restored in thevessel containing the dry powder. This was left to stand in air for afew seconds, after which it was used to make a 10% by weight aqueoussolution which showed under the microscope to be a suspension of verytiny bubbles (below 10 μm); the bubble concentration was in the range of10⁷ bubbles per ml. This preparation gave a very strong response inultrasonic echography at 2.25, 3.5, 5 and 7.5 MHz.

EXAMPLE 8

Two-dimensional echocardiography was performed in an experimental dogfollowing peripheral vein injection of 0.1-2 ml of the preparationobtained in Example 4. Opacification of the left heart with clearoutlining of the endocardium was observed, thereby confirming that themicrobubbles (or at least a significant part of them) were able to crossthe pulmonary capillary circulation.

EXAMPLE 9

A phospholipid/maltose lyophilized powder was prepared as described inExample 4. However, at the end of the lyophilization step, a ¹³³ Xecontaining gas mixture was introduced in the evacuated container insteadof air. A few minutes later, sterile water was introduced and aftergentle mixing a microbubble suspension containing ¹³³ Xe in the gasphase was produced. This microbubble suspension was injected into livingbodies to undertake investigations requiring use of ¹³³ Xe as tracer.Excellent results were obtained.

EXAMPLE 10 (COMPARATIVE)

In U.S. Pat. No. 4,900,540, Ryan et al disclose gas filled liposomes forultrasonic investigations. According to the citation, liposomes areformed by conventional means but with the addition of a gas or gasprecursor in the aqueous composition forming the liposome core (col. 2,lines 15-27).

Using a gas precursor (bicarbonate) is detailed in Examples 1 and 2 ofthe reference. Using an aqueous carrier with an added gas forencapsulating the gas in the liposomes (not examplified by Ryan et al)will require that the gas be in the form of very small bubbles, i.e. ofsize similar or smaller than the size of the liposome vesicles.

Aqueous media in which air can be entrapped in the form of very smallbubbles (2.5-5 μm) are disclosed in H. W. Keller et al, J. UltrasoundMed. 5 (1986), 413-498.

A quantity of 126 mg of egg lecithin and 27 mg of cholesterol weredissolved in 9 ml of chloroform in a 200 ml round bottom flask. Thesolution of lipids was evaporated to dryness on a Rotavapor whereby afilm of the lipids was formed on the walls of the flask. A 10 ml of a50% by weight aqueous dextrose solution was sonicated for 5 minaccording to M. W. Keller et al (ibid) to generate air microbubblestherein and the sonicated solution was added to the flask containing thefilm of lipid, whereby hand agitation of the vessel resulted intohydration of the phospholipids and formation of multilamellar liposomeswithin the bubbles containing carrier liquid.

After standing for a while, the resulting liposome suspension wassubjected to centrifugation under 5000 g for 15 min to remove from thecarrier the air not entrapped in the vesicles. It was also expected thatduring centrifugation, the air filled liposomes would segregate to thesurface by buoyancy.

After centrifugation the tubes were examined and showed a bottom residueconsisting of agglomerated dextrose filled liposomes and a clearsupernatant liquid with substantially no bubble left. The quantity ofair filled liposomes having risen by buoyancy was negligibly small andcould not be ascertained.

EXAMPLE 11 (COMPARATIVE)

An injectable contrast composition was prepared according to Ryan (U.S.Pat. No. 4,900,540, col. 3, Example 1). Egg lecithin (126 mg) andcholesterol (27 mg) were dissolved in 9 ml of diethylether. To thesolution were added 3 ml of 0.2 molar aqueous bicarbonate and theresulting two phase systems was sonicated until becoming homogeneous.The mixture was evaporated in a Rotavapor apparatus and 3 ml of 0.2molar aqueous bicarbonate were added.

A 1 ml portion of the liposome suspension was injected into the jugularvein of an experimental rabbit, the animal being under condition forheart ultrasonic imaging-using an Acuson 128-XP5 ultrasonic imager (7.5transducer probe for imaging the heart). The probe provided across-sectional image of the right and left ventricles (mid-papillarymuscle). After injection, a light and transient (a few seconds) increasein the outline of the right ventricle was observed. The effect washowever much inferior to the effect observed using the preparation ofExample 4. No improvement of the imaging of the left ventricle was notedwhich probably indicates that the CO₂ loaded liposomes did not pass thepulmonary capillaries barrier.

We claim:
 1. A dry pulverulent formulation which, upon dissolution inwater, will form an aqueous suspension of stabilized air or gasmicrobubbles useful in ultrasonic imaging, the formulation comprising atleast one film forming surfactant and at least one hydrophilicstabilizer in the presence of air or other entrappable gas, the filmforming surfactant being a saturated phospholipid in lamellar or laminarform.
 2. The dry formulation of claim 1, wherein the phospholipid isphosphatidic acid.
 3. The dry formulation of claim 1, wherein thephospholipid is phosphatidylcholine.
 4. The dry formulation of claim 1,wherein the phospholipid is phosphatidylethanolamine.
 5. The dryformulation of claim 1, wherein the phospholipid is phosphatidylserine.6. The dry formulation of claim 1, wherein the phospholipid isphosphatidylglycerol.
 7. The dry formulation of claim 1, wherein thephospholipid is phosphatidylinositol.
 8. The dry formulation of claim 1,wherein the phospholipid is cardiolipin.
 9. The dry formulation of claim1, wherein the phospholipid is sphyngomyelin.
 10. The dry formulation ofclaim 1, wherein the formulation further contains dicetylphosphate. 11.The dry formulation of claim 1, wherein the formulation further containscholesterol.
 12. The dry formulation of claim 1, wherein the formulationfurther contains ergosterol.
 13. The dry formulation of claim 1, whereinthe formulation further contains phytosterol.
 14. The dry formulation ofclaim 1, wherein the formulation further contains sitosterol.
 15. Thedry formulation of claim 1, wherein the formulation further containslanosterol.
 16. The dry formulation of claim 1, wherein the formulationfurther contains tocopterol.
 17. The dry formulation of claim 1, whereinthe formulation further contains propyl gallate.
 18. The dry formulationof claim 1, wherein the formulation further contains ascorbyl palmitate.19. The dry formulation of claim 1, wherein the formulation furthercontains butylated hydroxy-toluene.
 20. The dry formulation of claim 1,further containing dissolved viscosity enhancers or stabilizers in aweight ratio to the surfactants comprised between about 1:5 to 100:1.21. The dry formulation of claim 20, wherein the viscosity enhancers arelinear and cross-linked polysaccharides.
 22. The dry formulation ofclaim 20, wherein the viscosity enhancers are linear and cross-linkedoligo-saccharides.
 23. The dry formulation of claim 20, wherein theviscosity enhancers are sugars or hydrophilic polymers.
 24. The dryformulation of claim 1, wherein the formulation comprises up to 50% byweight of non-laminar surfactants.
 25. The dry formulation of claim 24,wherein the non-laminar surfactant is a fatty acid.
 26. The dryformulation of claim 24, wherein the non-laminar surfactant is an esteror ether of fatty acids and alcohols.
 27. The dry formulation of claim24, wherein the non-laminar surfactant is a polyalkylene glycols. 28.The dry formulation of claim 24, wherein the non-laminar surfactant is acarbohydrate.
 29. The dry formulation of claim 24, wherein thenon-laminar surfactant is a polyalkylenated glycerol.
 30. The dryformulation of claim 1, wherein the phospholipids in laminar form are inthe form of fine layers deposited on the surface of soluble or insolublesolid particulate material.
 31. The dry formulation of claim 30, whereinthe insoluble solid particulate material is glass or polymer beads. 32.The dry formulation of claim 30, wherein the soluble particles are madeof hydrosoluble carbohydrates or polysaccharides.
 33. The dryformulation of claim 30, wherein the soluble particle are made ofsynthetic polymers or Iopamidol.
 34. The dry formulation of claim 30,wherein the soluble particles are made of albumin or gelatin.
 35. Thedry formulation of claim 1, in the form of freeze-dried liposomes. 36.The dry formulation of claim 1, wherein the size of a majority of themicrobubbles is below 50 μm.
 37. The dry formulation of claim 1, whereinthe suspension contains about 10⁸ to 10⁹ bubbles of 0.5 to 10 μmsize/ml.
 38. The dry formulation of claim 1, wherein the entrappable gasis a freon, nitrogen, carbon dioxide, argon, methane or mixturesthereof.
 39. The dry formulation of claim 1, wherein the entrappable gasis a freon.
 40. The dry formulation of claim 1, wherein the entrappablegas is ⁸¹ kripton or ¹³³ xeonon.
 41. The dry formulation of claim 38,wherein the entrappable gas is a halogenated hydrocarbon gas.
 42. Thedry formulation of claim 1, wherein the entrappable gas is nitrogen. 43.The dry formulation of claim 1, wherein the entrappable gas is carbondioxide.
 44. The dry formulation of claim 1, wherein the entrappable gasis argon.
 45. The dry formulation of claim 1, wherein the entrappablegas is methane.
 46. The dry formulation of claim 1, wherein thehydrophilic stabilizer is a hydrosoluble protein.
 47. The dryformulation of claim 1, wherein the hydrophilic stabilizer is apolypeptide.
 48. The dry formulation of claim 1, wherein the hydrophilicstabilizer is a sugar.
 49. The dry formulation of claim 1, wherein thehydrophilic stabilizer is a poly- or oligo-saccharide.
 50. The dryformulation of claim 1, wherein the hydrophilic stabilizer is ahydrophilic polymer.